Free radical and reactive oxygen species (ROS) play important roles in the etiology and/or progression of a number of diseases and in aging as well as in signal transduction. Investigators in the Section on Metabolic Regulation carried out studies to elucidate mechanisms by which free radicals and ROS are generated and exert their biological effects. During this fiscal year, we continued to focus on the roles of gluationylation of actin and protein tyrosine phospatase 1B induced by ROS cell signaling. The investigation reveals that both actin and protein tyrosine phosphatase 1B are superior substrates for glutareodoxin-facilitated glutathionylation with glutathione-thiyl radical. Furthermore, glutaredoxin also catalyzed the deglutathionylation of glutathionylated actin. RNA interference was employed to knockdown glutaredoxin expression. To overcome various shortcomings of this method when applied to mammalian cells, a vector was developed to have tetracycline regulate the synthesis of full-length double-stranded RNA for glutaredoxin in mammalian cells. This method has proven to be successful at the cellular level and it is now being extended to establish transgenic mice with the tetracycline-controllable dsRNA-generating gene to ensure that protein knockdown can be reversibly performed at any stage of animal development. We are currently applying this method to investigate a number of redox enzymes. Mechanistic studies are also being carried out for post-transcriptional interference. To this end, we have isolated a protein that likely participates in the RNAi pathway. In addition, hydrogen peroxide was used to study the response of B cells to oxidative stress with respect to phosphatidylinositol 3-kinase (PI3K)/Akt activation pathway. We previously showed that Bruton?s tyrosine kinase (Btk) deficiency did not inhibit but rather it potentiated hydrogen peroxide-induced activation of PI3K and Akt. Current results show that this activation is mediated by PKCmu activation induced by hydrogen peroxide in DT40 cells and leads to upregulation of Syk activity required for the tyrosine phosphorylation of P110alpha, the catalytic subunit of PI3K, and its associated 98 Kda protein. This feedback regulation of PI3K/Akt activation by Btk appears to be agonist specific, i.e. induction by hydrogen peroxide vs. anti-IgM. Earlier we showed that serum deprivation leads to elevation of ROS levels and apoptosis in neuroblastoma SH-SY5Y cells. Hormesis and anti-apoptotic effects were also observed. These effects are mediated by the cyclic GMP-dependent protein kinase pathway that leads to elevated expression of thioredoxin, Mn-superoxide dismutase, and anti-apoptotic Bcl-2. This protective mechanism was found to be responsible for the neuroprotective action of selegiline against oxidative stress and apoptosis caused by 1-methyl-4-phenyl-pyridinium in the 1-methyl-4-phenyl-1,2,36-tetrahydropyridine-induced Parkinsonian animals.